Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/04—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from milk

Definitions

• the present invention relates to a process for the separation of immunoglobulins from colostrum.
• Colustrum is the mammary secretion of female mammals, secreted during the days that follow parturition. Its composition and appearance are very different from those of milk and remain so until the seventh day. Its characteristics are, among others, richness in proteins, especially in immunoglobulins of which the content varies from 6 to 10% on first milking to 0.09% which is the normal content of milk. These proteins, and especially the immunoglobulins, are of great interest, especially in the medical and pharmaceutical field. They can, in particular, be added to foods of new-born calves and serve to increase their disease immunity, or may be used in medicine, pharmaceuticals or human foodstuffs and particularly for children's foods.
• a process for separating proteins by means of ion-exchange chromatography is disclosed in U.S. Pat. No. 4,100,149.
• Ion-exchange materials are used, for this purpose, consisting of porous particles on the surface of which are grafted ion-exchange groups.
• This patent describes the separation of immunoglobulins from human serum from which lipids have been removed on an anionic exchanger material (consisting of particles of silica coated with a polymer having anionic sites), the immunoglobulins being recovered in the effluent "in an electrophoretically pure state" while the other proteins are fixed.
• the Applicant has found that it was possible to separate the immunoglobulins present in colostrum if the colustrum milk or colostrum serum is fractioned by electrophoresis before carrying out the ion-exchange chromatography.
• this invention relates to a process for the separation of the immunoglobulins present in colostrum wherein a milk of colostrum or a serum of colostrum is fractioned by liquid electrophoresis, the immunoglobulin-enriched fraction is recovered and the said immunoglobulin-enriched fraction is then fractioned by ion-exchange chromatography.
• the raw material used is, preferably, colostrum from cows but may be from any other mammal.
• Fractionation is not performed on natural colostrum but on milk of colostrum from which the cream has been removed or, preferably, on serum of colostrum which has been subject to skimming and coagulation in order to remove the casein.
• Said coagulation may be carried out by adding rennet of strength 1/10,000 on the basis of about 0.1 ml/l, or by acidic precipitation.
• the first fractionation used in the present invention is fractionation by liquid electrophoresis.
• This type of fractionation generally consists of fractionating a solution comprising at least two groups of substances dissolved in a liquid in order to obtain at least two liquid fractions one of which contains the first group of substances with a relative richness higher than that of the original solution and the other contains the second group with a relative richness higher than that of the said solution; for this purpose, the initial solution is transferred into at least one fractionation chamber limited by two semi-permeable membranes on two principal opposing sides and an electric field is applied to the said chamber so as to cause migration of the groups of substances in relation to their electrical and physical characteristics, causing natural convection by the effect of concentration gradients with the creation of ascending and descending flows of liquid, and the liquid is withdrawn from at least two different zones in order to collect said fractions.
• Fractionation may, in particular, be performed in accordance with the process described in patent No. FR 2 493 725 and the Doctor of Engineering Thesis of the Faculty of Science of Toulouse: preparation of proteinaceous fractions of serum or of blood plasma by electrophoresis, P. Espenan, 1980.
• An electric field is used in this process to provide through at least one of the semi-permeable membranes, a high-speed ion-exchange between the original solution contained in the fractionation chamber or chambers and an auxiliary solution placed on the other side of the said membrane and containing ions of the type suitable to pass through the said membrane and to increase the differences of migration speed of the substances from the original solution.
• the original solution is admitted into a series of fractionation chambers that are side-by-side and separated from each other by the semi-permeable membranes, while the auxiliary solution is placed either side of the two end chambers in two storage compartments each of which is separated by a semi-permeable membrane from the neighboring fractionation chamber and ion-exchange is performed at high speed under the effect of the electrical field, from the said storage compartments through the fractionation chambers, from one to the next.
• This process thus provides a means of providing, in one operation and in the same device, an adaptation of the original solution to give it the appropriate properties needed to accentuate the differences of migration of the substances, firstly, and the specific operation of fractionating the said substances, secondly.
• the electric field used for fractionation also serve to generate rapid ion-exchange through the membranes between the original solution contained in the fractionation chambers and the auxiliary solution contained in the storage compartments. Operations of preliminary preparation are thus radically eliminated, especially the dialysis that is required in traditional processes and also the corresponding disadvantages; moreover, the original solution no longer has to be diluted and the process can be applied to natural solutions in the state in which they are produced or collected.
• This process has already been used for fractionation and recovery of plasma immunoglobulins in order to separate an albumin-rich fraction and a fraction rich in ⁇ -globulins.
• the separation by liquid electrophoresis in the present invention is performed advantageously at a pH of 6 to 7, preferably 6.3-6.4 (that is, in practice generally with no modification of pH) and at a temperature of 4° C. to 25° C., preferably 4° C.
• a fraction that is relatively richer at the nature state, in immunoglobulins than the original concentration is recovered at the top of the electrophoresis device and a fraction relatively richer in albumins at the bottom.
• composition in percent weight, respectively, in the fractions relatively richer in immunoglobulins and in albumins is obtained.
• the quantity of immunoglobulins recovered at the top of the device depends on operating conditions and, among others, on the rate of withdrawal; it can reach 70 to 80%.
• the second fractionation process used in the present invention is fractionation by ion-exchange chromatography. It consists of submitting the fraction rich in immunoglobulins obtained by the first fractionation process, to ion-exchange chromatography.
• the immunoglobulin-rich fraction is precolated, for this purpose, through a column filled with ion-exchange particles.
• Spherosils Q MA, C and S are obtained by fixing ion-exchanger resins on beads of porous silica (X OB 0 15).
• ion-exchanger that provides selective fixing of proteins
• grafted celluloses such as the anion-exchanger resins "Trisacryl” sold by I.B.F.
• Anionic exchangers are used preferably, such as Spherosil Q MA exchangers that provide a means of directly treating the immunoglobulin-rich fraction obtained during the first stage, without modifying the pH(between 6 and 7) and obtaining a high yield of immunoglobulins in the effluent that leaves the column. This affords the advantage of preventing any risk of denaturation of the immunoglobulins by fixation on the ion-exchangers.
• a solution of proteins can thus be obtained containing 98 to 100% of immunoglobulins.
• the chromatography can be performed at a temperature from 4° to 25° C. and, advantageously, at 4° C.
• the solution of immunoglobulins can then be post-treated, by drying, concentration, dialysis or ultrafiltration for the purpose of later use, for example.
• This colostrum serum is fractionated by electrophoresis using the device described in application No. FR 2 493 725 at a temperature of 4° C.
• This device comprises five fractionation chambers separated by semi-permeable cellophane membranes of which the active surface of each is 60 cm 2 .
• Each chamber is 3 mm thick and comprises a frame that divides it into eleven vertical channels against which the membranes bear.
• the auxiliary solution circulating in the side storage compartments is a mixture of Na 2 HPO 4 ; and KH 2 PO 4 ; at a saline concentration of 0.05M and at a pH of 6.25.
• a solution of Na 2 HPO 4 and KH 2 PO 4 also circulates in the ajoining electrode compartments but at a concentration of 0.2M.
• the quantity of immunoglobulins obtained at the device top depends on experimental conditions and approaches 80% in the third test.
• the effluent only contains immunoglobulins.
• the electroimmunodiffusion analysis moreover indicates that they are exclusively IgG 1 and, more specifically, the fraction of the IgG 1 's with the highest isoelectric point. This fraction should also contain the lactotransferrins of which the isoelectric point is 7.8
• the albumins are recovered by elution with 0.1N HCl, as are the other immunoglobulins, IgG 1 , IgA abd IgM.
• the average fixation capacity of the Q MA Spherosil is, in the present case, 110 mg/g.
• This colostrum serum is percolated directly through a column of Spherosil Q MA ion-exchanger containing 64 g of resin (identical to that used in example 1).
• the fixation capacity is 96 mg/g at pH 6.3 and at 4° C. with flowrate 300 ml/h.
• compositions of the effluent and of the eluate obtained with a 0.1N solution of HCl are as follows:

Landscapes

• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Health & Medical Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Biochemistry (AREA)
• Biophysics (AREA)
• Life Sciences & Earth Sciences (AREA)
• Genetics & Genomics (AREA)
• Medicinal Chemistry (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Immunology (AREA)
• Peptides Or Proteins (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=9270352

Family Applications (1)

Country Status (6)

Cited By (20)

Families Citing this family (4)

Citations (8)

• 1982
  • 1982-01-27 FR FR8201230A patent/FR2520235A1/fr active Granted
• 1983
  • 1983-01-20 EP EP83400141A patent/EP0085005B1/de not_active Expired
  • 1983-01-20 AT AT83400141T patent/ATE17358T1/de not_active IP Right Cessation
  • 1983-01-20 DE DE8383400141T patent/DE3361733D1/de not_active Expired
  • 1983-01-25 US US06/460,905 patent/US4582580A/en not_active Expired - Fee Related
  • 1983-01-26 CA CA000420316A patent/CA1196310A/en not_active Expired

Patent Citations (8)

Non-Patent Citations (8)

Also Published As

Similar Documents

Legal Events